Regulation of 1,25-Dihydroxyvitamin D3 Receptor Gene Expressionby 1 ,25-Dihydroxyvitamin D3 in the Parathyroid In VivoTally Naveh-Many, Ruth Marx, Eli Keshet, J. Wesley Pike, and Justin SilverMineral Metabolism Unit, Nephrology Services, and the Department of Virology, Hadassah University Hospital, Jerusalem, il-91120,Israel; and the Department ofPediatrics and Endocrinology, Baylor College ofMedicine, Texas Medical Center, Houston, Texas 77030

Abstract

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) dramatically de-creases parathyroid hormone (PTH) gene transcription. Wehave now studied the effect of 1,25(OH)2D3 on the1,25(OH)2D receptor (VDR) in the parathyroid in vivo. Ratswere injected with 1,25(OH)2D3 and the parathyroid-thy-roid tissue analyzed for PTHmRNA and VDRmRNA.1,25(OH)2D3 (50 and 100 pmol ip) decreased PTHmRNA at 6h with a maximum at 48 h (< 4% of basal), whereasVDRmRNA was increased only after 6 h with a 1.7-fold in-crease at 24 h. VDRmRNA levels peaked at 25 pmol1,25(OH)2D3 with a twofold increase. Serum calcium did notaffect VDRmRNA. Parathyroid VDRmRNA ran at 2.2 and 4.4kb, whereas duodenum VDRmRNA had a single band, all ofwhich increased after 1,25(OH)2D3. Weanling rats on a vita-min D-deficient diet for 3 wk had a more intense 2.2-kb tran-script, whereas vitamin D-replete rats had a more intense 4.4-kb band.

Dispersed parathyroid-thyroid cells were separated by a

flow cytometry (FACS) into a parathyroid cell peak containingPTHmRNA and a second peak with cells positive for thyro-globulin mRNA and calcitonin mRNA. VDRmRNA was con-

centrated in the parathyroid cell peak. In situ hybridization ofparathyroid-thyroid and duodenum for VDRmRNA showed itslocalization to the parathyroid cells and the duodenal mucosa.

Therefore, the VDRmRNA in the parathyroid-thyroid tissuerepresents predominantly parathyroid cell and not C-cellVDRmRNA which is also a 1,25(OH)2D3 target organ. Theincreased VDR gene expression in the parathyroid cell wouldamplify the effect of 1,25(OH)2D3 to decrease PTH gene tran-scription. (J. Clin. Invest. 1990. 86:1968-1975.) Key words:calcium * flow cytometry * in situ hybridization * calcitonintranscription

Introduction

The vitamin D sterol's biologically active metabolite 1,25 di-hydroxyvitamin D3 (1,25(OH)2D3) regulates mineral homeo-stasis by acting on its classical target organs, the intestine,bone, and kidney (1, 2). It also regulates mineral homeostasisby decreasing the transcription of the calciotrophic hormones,

Address reprint requests to Dr. J. Silver, Mineral Metabolism Unit,Nephrology Services, Hadassah University Hospital, POB 12000, Je-rusalem, Israel il-9 1120.

Receivedfor publication 4 June 1990 and in revisedform 30 July1990.

parathyroid hormone (PTH)' (3-5) and calcitonin (6, 7).1 ,25(OH)2D3 acts on its diverse target tissues by binding to the1,25(OH)2D receptor (VDR), a 48-kD protein which has beencloned, and shown to be structurally similar to other steroidreceptors (8). The DNA sequences in the rat and human os-teocalcin genes that bind the 1,25(OH)2D3 receptor and conferresponsiveness to 1,25(OH)2D3 have now been determined(9-1 1), thus providing further evidence that 1,25(OH)2D3binds to its receptor which activates gene transcription analo-gous to that of other small nonpeptide hormones (12). Muta-tions in the VDR gene result in 1,25(OH)2D3-resistant rickets,further confirming the importance of the VDR (13, 14).

The VDR concentration in the 1,25(OH)2D3 target siteswould allow a modulation of the 1 ,25(OH)2D3 effect. Ligand-dependent upregulation of the VDR has been shown in vivo inrat intestine (15-17) at the levels of VDRmRNA and VDRprotein content (18). In vitro ligand-dependent upregulationhas been shown on VDRmRNA in 3T6 cells (8), human os-teosarcoma cell lines (19), and human leukemia cells (HL 60)(20, 21).

We have shown previously that in vivo in the rat1,25(OH)2D3 dramatically decreased PTH gene transcription(4), and we now demonstrate that this is associated with anupregulation of the VDRmRNA levels in the parathyroids.

Dot blots of RNA extracts of parathyroid-thyroid tissuefrom normal rats which had been hybridized with PTHcDNAand actin cDNA were now washed and rehybridized forVDRmRNA. Other rats were injected with 1,25(OH)2D3 andtheir parathyroid-thyroid extracts run on agarose gels forNorthern blots for PTHmRNA and VDRmRNA. These re-sults were supplemented by studies which demonstrated thecellular localization of the VDRmRNA to the parathyroids.

Methods

Animals. Male Hebrew University (Jerusalem, Israel) strain rats(150-180 g) were maintained on a normal diet and injected ip withvitamin D metabolites dissolved in propylene glycol (100 ,u), or with0.6 M sodium phosphate monobasic (0.2-2 mmol/100 g body wt),calcium gluconate lactate (2 ml), or calcitonin (synthetic human calci-tonin, CIBA-Geigy, Basel, Switzerland) as previously described (4,6, 22).

In one series of experiments weanling male rats were housed in anultraviolet free environment for 3 or 8 wk and fed ad libitum one ofthefollowing diets (Teklad, Madison, WI): normal vitamin D, 0.4% cal-cium (NDNCa) (TD 88304); normal vitamin D, 0.02% calcium(ND-Ca) (TD 88346); vitamin D deficient, 0.4% calcium (-DNCa)(TD 85049); vitamin D deficient, 0.02% calcium (-D-Ca) (TD85048); vitamin D deficient, 2% calcium (-D+Ca) (TD 89155).

At timed intervals the parathyroid-thyroid tissue was excised under

1. Abbreviations used in this paper: PTH, parathyroid hormone; VDR,1,25(OH)2D receptor.

1968 Naveh-Many, Marx, Keshet, Pike, and Silver

J. Clin. Invest.© The American Society for Clinical Investigation, Inc.0021-9738/90/12/1968/08 $2.00Volume 86, December 1990, 1968-1975

pentobarbital anesthesia, and blood samples taken. In some experi-ments also duodenum was excised (first 10 cm) after flushing withice-cold PBS, and the mucosa was separated from the serosa with aglass microscope slide on ice. The excised tissue was flash frozen inliquid nitrogen and stored at -70°C until extraction. Serum calciumwas measured with an atomic absorption spectrophotometer (Perkin-Elmer Corp., Pomona, CA).

Measurement of cellular mRNA levels. Total cellular RNA wasprepared from frozen tissue by homogenization in guanidium thiocya-nate followed by deproteinization using guanidine hydrochloric acidand ethanol precipitation as previously described (4). For the dot blothybridization assay, total tissue RNA extracts were prepared by form-aldehyde denaturation followed by serial dilutions with 15X SSC (I X= 0.15 M NaCl, 0.01 M Na citrate), and blotted onto nitrocellulosefilters (0.45 Am; Schleicher& Schuell, Keene, NH) soaked in lOx SSC,using a manifold apparatus (Bethesda Research Laboratories, Gaith-ersburg, MD). For Northern blot analysis RNA and markers werefractionated under denaturing conditions on a 1.25 or 1.5% formalde-hyde-agarose gel and transferred to nitrocellulose filters by diffusionblotting. The mRNA size was determined from the migration of 28Sand 18S ribosomal RNA. The filters were baked at 80°C for 120 min ina vacuum oven.

Hybridization. An 833-bp rat preproPTHcDNA was obtained byrestriction enzyme (Kpn I, Pst I) digestion of plasmid PT 43, andlabeled by nick translation to a specific activity of 2-5 X 108 cpm.Hybridization was carried out over night at 42°C, autoradiographed,and the films scanned with a densitometer.

I,25(OH)2D3 receptor mRNA levels were measured by hybridiza-tion with the human 1,25(OH)2D3 receptor cDNA (2.1-kb insert)cloned in the p GEM 7 Z f (+) vector (23). The plasmid serves as astandard cloning vector and a template for in vitro transcription ofthereceptor mRNA which is labeled to a high-specific activity RNAprobe. A modification of the method of Kreig et al. (24) was used asfollows. 100 ng Hind III-linearized template DNA was used in 10 Mlreaction volume. An incubation of 10mM DTT, 500MM each ofATP,CTP, GTP, 3,000 Ci/mmol 32P-UTP, 40 mM Tris HCI, pH 7.5, 60mM MgCl2, 2 mM spermidine-HCl, 100 U/ml T 7 polymerase, 1U/ml human placental ribonuclease inhibitor was carried out at 37°Cfor 20 min. The DNA template was removed by the addition of 1 Mlribonuclease-free DNase (1 U/Mg DNA) and incubated at 37°C for 10min. The reaction mix was diluted with water to a total volume of 100Ml. The specific activity of the RNA transcript was - 6 x 108 cpm/Mg.5- 10 Ml was used for each 1 ml hybridization mix.

For in situ hybridization the anti-sense VDR RNA probe was syn-thesized, by linearizing the p GEM 7 Zf (+) plasmid with Hind IIIdigestion. [35S]-labeled RNA was synthesized in the antisense orienta-tion as described above using the T7 polymerase. Before in situ hybrid-ization the probe was fragmented by a mild alkaline treatment to100-200-bp fragments.

Flow cytometry ofthyroparathyroid cells. Thyroparathyroid tissuewas minced in 5% FCS at 4°C, and then incubated with collagenaseCLSI (500 U/ml) (Worthington Diagnostics, Freehold, NJ) at 37°Cfor 1 h in a shaking water bath. The glands were digested completelyinto isolated cells. The cell suspension was filtered through a nylonmesh, centrifuged at 400 g for 5 min at 4°C, washed twice in HBSS andthe pellet resuspended in 500 Ml of HBSS. The membrane integrity ofthe cell suspension was demonstrated by incubating the cells with3',6'-diacetyl fluorescein (FDA) (Sigma Chemical Co., St. Louis, MO)(1 Ml per 100-Mul cell suspension). Intact cells have nonspecific esteraseswhich cleave the FDA, which readily crosses cell membranes, into freefluorescein which is highly fluorescent (24-26). 80% ofthe cell suspen-sion exhibited positive fluorescence. The cell suspension was analyzedby a FACS 440 cell sorter (Becton-Dickinson & Co., Paramus, NJ)using 300 mV excitation at 488 nm from an argon ion laser source.The diameter ofthe nozzle used was 70Mum. The fluorescent angle lightscatter was at 520 nm. The integrity of the cells collected from the cellsorter have also been demonstrated by propidium iodide staining(> 90% intact cells) and by microscopy of cells after concentration and

staining with Papanicoula stain (27). In some experiments RNA wasextracted from the cells collected from the cell sorter and analyzed formRNA content as before.

In situ hybridization analysis. Sections of parathyroid-thyroid andduodenum were prepared for in situ hybridization and hybridized asdescribed by Hogan et al. (28). Briefly, 10-Mm-thick frozen sectionswere collected on poly-L-lysine-coated glass slides, refixed in 4% para-formaldehyde, and dehydrated in graded ethanol solutions. Beforehybridization sections were pretreated successively with 0.2 N HCI, 2xSSC, 0.125 mg/liter pronase, 4% paraformaldehyde, and acetic anhy-dride in triethanolamine buffer. Hybridization was carried out at 50°Covernight in 50% formamide, 0.3 M NaCl containing 10% dextransulfate, I X Denhardt's solution, I mg/ml carrier tRNA, 10 mM DTT,5 mM EDTA, and 2X 108 cpm/ml 3"S-labeled 1,25(OH)2D3 receptorriboprobe. Organs were immediately fixed in 4% paraformaldehyde inPBS for 16 h at 40C, then incubated for 24 h in 0.5 M sucrose in PBS,after which they were embedded in OCT embedding medium (MilesScientific Inc., Naperville, IL) and frozen. Posthybridization washingwas performed under stringent conditions that included an incubationat 50°C for > 4 h in 50% formamide/0.3 M NaCl and a 30-min incu-bation at 37°C in 20 Mg/ml RNase A (28, 29). Autoradiography wasperformed using Kodak NTB-2 nuclear track emulsion.

Results

After a single injection of 1,25(OH)2D3 (50 or 100 pmol) tonormal rats we had demonstrated a decrease in PTHmRNAlevels at 6 h, reaching < 4% of basal at 48 h after 100 pmol1,25(OH)2D3 (4). These same filters were extensively washedand then rehybridized with the VDR riboprobe (Fig. 1). At 6 hafter both 50 and 100 pmol 1,25(OH)2D3 VDRmRNA levelsin these rat parathyroid-thyroid RNA extracts were less butnot significantly different from basal levels. At 24 h there was a

200

E VDR

100

1OOpmol

0 10 20 30 40 50

TIME (hours)Figure 1. Time course of the effect of 1,25(OH)2D3 on mRNA levelsfor parathyroid hormone (PTH) and the 1,25(OH)2D3 receptor(VDR) in rat parathyroid-thyroid tissue. Rats were injected with ei-ther a single dose of 100 pmol 1,25(OH)2D3, or 50 pmol1,25(OH)2D3 at 0 and 24 h. The arrow represents the second injec-tion of 50 pmol 1,25(OH)2D3. Levels ofmRNA for PTH (*, .) andVDR (o, o) were determined by sequential hybridizations. The datafor PTHmRNA has been published previously (4). The results ofeach point are the mean±SE for four rats and are expressed as a per-centage of basal.

1,25(OH)2D Receptor Gene Expression 1969

1.7-fold increase in VDRmRNA at both doses. Rats given asingle dose of 100 pmol 1,25(OH)2D3 had a decrease inVDRmRNA at 48 h, whereas those given 50 pmol1,25(OH)2D3 had received a second 50-pmol dose at 24 h andthey maintained their elevated VDRmRNA levels for 48 h.Actin mRNA was not affected by 1,25(OH)2D3 (data notshown). This time course of 1,25(OH)2D3 on VDRmRNA in-dicates an effect after 6 h, evident at 24 h but not at 48 h after asingle dose.

A 1,25(OH)2D3 dose response study at 24 h had shown adecrease in PTHmRNA levels after as little as 12.5 pmol withlittle change from 50 to 200 pmol (4). Actin mRNA was notaffected. This data is shown in Fig. 2 together with results afterrehybridization with VDR riboprobe to measure VDRmRNAlevels. VDRmRNA levels were increased after 12.5 pmolpeaking at 25 pmol with a twofold increase. These doses of1,25(OH)2D3 had not caused hypercalcemia.A Northern blot of parathyroid-thyroid tissue from rats

injected with 1,25(OH)2D3 (100 pmol at 24 h) demonstrated areduction in PTHmRNA which ran as a single band at 833 bp(Fig. 3). Hybridization for VDRmRNA showed 2 bands at 2.2and 4.4 kb in the control rats, and both bands were increasedby a single injection of 1 ,25(OH)2D3 (100 pmol at 24 h). RNAextracts of duodenum were run on agarose gels and Northernblots for VDRmRNA showed a single band at 4.4 kb for acontrol rat. After 1,25(OH)2D3 (100 pmol at 24 h) there was anincrease in intensity of this single band (Fig. 4).

In vivo in the rat a low calcium increased PTHmRNAlevels at 1-6 h (22). Rehybridization of those filters showed noeffect of serum calcium on VDRmRNA levels (Fig. 5).

Chronic changes in dietary calcium and vitamin D regulatePTHmRNA levels in the rat. We therefore studied rats whohad been maintained for 3 wk after weaning on different diets.The diets used were either vitamin D normal (ND) or deficient(-D), together with different calcium concentrations: 0.02%(-Ca), 0.4% (NCa), 2% (+Ca).

The mean serum calciums in the different groups of ratswere NDNCa, 10.6 mg/dl; ND-Ca, 6.3 mg/dl; ND+Ca, 12.2mg/dl; -DNCa, 10.6 mg/dl; -D-Ca, 5.1 mg/dl; -D+Ca, 11.2mg/dl. We had shown in previous studies that the vitaminD-deficient diet given for 3 wk to weanling rats did not resultin low serum 1,25(OH)2D levels in -DNCa rats, but only in-D-Ca rats, whereas ND-Ca rats had markedly increasedlevels of serum 1,25(OH)2D (27).

Northern blot hybridization showed that PTHmRNA ran

Figure 2. 1,25(OH)2D3dose-response effect on

200 mRNA levels for para-thyroid hormone (PTH)

VDR (o) and the1 ,25(OH)2D3 receptor

EC (VDR) (o), in rat para-100.\ thyroid-thyroid tissue

> loo-11 24 h after administra-tion of 1,25(OH)2D3 ip.

z \ The data forE PTH PTHmRNA has been

published previously0 (4). The results repre-0 100 200 sent mean±SEM for

1.25(01D)2D3 (pmol) four rats.

VDR

..28S

18S

Figure 3. Gel blot anal-ysis of total RNA from

PTH parathyroid-thyroid tis-sue. Filters were hybrid-

3 4 ized sequentially forPTHmRNA andVDRmRNA. Lanes: Iand 3, parathyroid-thy-roid tissue of one con-trol rat; 2 and 4, 1 rat24 h after 100 pmol1,25(OH)2D3. RNA sizeis shown for ribosomal18S and 28S RNAS, in-dicating the approxi-mate positions of the2.2 and 4.4 kb tran-script, respectively.

as a single band (833 bp) which was increased by calciumdeficiency (ND-Ca) even more so when combined with vita-min D deficiency (-D-Ca) (Fig. 6). A high calcium (-D+Ca)did not affect PTHmRNA mobility or decrease the intensity ofthe transcript. After extensive washing the Northern blot wasrehybridized for VDRmRNA. The VDRmRNA ran as twobands at 2.2 and 4.4 kb (Fig. 6). In control rats (NDNCa) andcalcium-deficient rats (ND-Ca) the predominant band was at4.4 kb (Fig. 6). Similar results were seen in ND+Ca rats (notshown). In contrast in rats after 3 wk of a vitamin D-deficientdiet, regardless of the calcium content of the diet (NCa, -Ca,+Ca), the predominant band was at 2.2 kb.

We have previously shown that separation of parathyroid-thyroid cells by enzymatic digestion and passage through aflow cytometer (FACS) separates parathyroid from thyroidcells (27). A parathyroid-thyroid cell suspension was passedthrough a FACS and the smaller parathyroid cells separatedand collected separately from the larger thyroid cells (Fig. 7).The RNA extracted from cells from each peak were hybridizedsequentially with PTHcDNA, thyroglobulin cDNA, calcitonincDNA, and VDR riboprobe (Fig. 7). PTHmRNA was onlypresent in the first peak (Fig. 7) and thyroglobulin mRNA inthe second peak (27). Moreover calcitonin mRNA was onlypresent in the second peak, whereas VDRmRNA was almostall in the first peak with a weak message in the second peak(Fig. 7). These results show that the VDRmRNA in the para-thyroid-thyroid tissue extracts was mainly derived from para-thyroid tissue.

We performed in situ hybridization with the VDR ribo-

0 6 24

4200

X- - Figure 4. Gel blot anal-2 3 4 5 6- ysis of total RNA from

duodenum hybridizedfor VDRmRNA. Lanes:

T-k,' . 1 and 2, control; 3 and~~~~~~~~4, 6 h afterI _25(OH)2D3 (100pmol);5 and 6, 24 h

dj.../ after 1,25(OH)2D3 (100pmol). RNA size isshown in nucleotidebase pairs.

1970 Naveh-Many, Marx, Keshet, Pike, and Silver

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DO Figure 5. The effect ofchanges in serum cal-

Doo cium from control ratsat 10.4 mg/dl on

00O PTH mRNA levels for PTHPTH (o) and VDR (*). The

oo- Wa results representmean±SEM for four

Doog srats as a percentageVDR of basal. The data

0 for PTHmRNA has0 10 20 been published pre-

SERUM CALCIUM (mg/di) viously (22).

probe. 35S-labeled antisense RNA was synthesized in vitro andused as an hybridization probe. 10 -,um-thick frozen sections ofparathyroid-thyroid tissue and proximal small intestine were

prepared from a control rat and a 1,25(OH)2D3 (100 pmol at24 h)-treated rat.

Bright field illumination of parathyroid-thyroid tissue (Fig.8 A) from a control rat (Fig. 8 A1) and a rat injected with1,25(OH)2D3 (100 pmol at 24 h) (Fig. 8 A2) demonstrated theparathyroid glands (white arrows) embedded in the thyroidtissue. Duodenum from a 1,25(OH)2D3-treated rat is demon-strated on the same preparation (Fig. 8 A3). A higher-powermagnification (Fig. 8 B) demonstrates the parathyroid gland(p) and thyroid follicles (t).

Sections were hybridized under conditions that favor for-mation of RNA-RNA hybrids (28, 29). VDRmRNA in bothcontrol and 1,25(OH)2D3-treated parathyroid-thyroid tissuewas localized to the parathyroid glands (Fig. 8 C). Backgroundactivity was present on the thyroid follicles with a small in-crease in the parafollicular cells. VDRmRNA was also presentin the rat proximal small intestine mucosal cells, both cryptand villi cells with none in the muscularis and serosa. The

VDR

1 2 3 4

PTH

12 3 4

-LJm

C-i

=:LL

PTH

CT

VDR

Figure 7. Flow cytometry analysis of a dispersed rat parathyroid-thy-roid cell suspension. The cells were analyzed by size and the cellsfrom the two peaks (0-110 nm and 115-200 nm) were collected sep-arately, the RNA extracted and then hybridized sequentially withcDNAs for PTH, thyroglobulin (Tg), and calcitonin, and the1,25(OH)2D3 receptor (VDR) riboprobe. The three dots in each row

are from increasing volumes of total RNA extracted from a collectedcell population. The second row is a duplicate.

28S

18S

Figure 6. Gel blot analysis of total RNA from parathyroid-thyroidtissue of rats maintained for 3 wk after weaning on diets containingnormal vitamin D (ND) (lanes I and 2) or no vitamin D (-D) (lanes3 and 4), together with 0.4% calcium (NCa) (lanes I and 4), or

0.02% calcium (-Ca) (lanes 2 and 3). Lane 1, NDNCa; lane 2,ND-Ca; lane 3, -D-Ca; lane 4, -DNCa. The filters were hybridizedsequentially for PTHmRNA and VDRmRNA. RNA size is shownfor ribosomal 1 8S and 28S RNAs, indicating the positions of the 2.2and 4.4 kb transcripts, respectively.

amount ofVDRmRNA in the parathyroids was similar to thatin the small intestine crypts (Fig. 8 C). In a rat injected with1,25(OH)2D3 (100 pmol at 24 h) there was an apparent in-crease in VDRmRNA in the parathyroids (Fig. 8) consistantwith the 1.7-fold increase shown in Fig. 1, but the in situ datawas not quantified. A higher power view of the parathyroid-thyroid tissue shown in Fig. 8 B, revealed the marked concen-

tration of the VDRmRNA in the parathyroid gland, as com-

pared to the thyroid tissue (Fig. 9 A), and in the intestinal villuscrypt cells (cr), and not in the muscularis (m) and serosal (s)layers (Fig. 9, B and D).

Discussion

1,25(OH)2D3 is a potent regulator of PTH gene transcription,leading to a large decrease in PTH gene transcription andPTHmRNA levels in vivo and in vitro (3-5). We have now

1,25(OH)2D Receptor Gene Expression 1971

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shown that 1,25(OH)2D3 leads to a dose-dependent increase inVDRmRNA levels in the parathyroid. The increase inVDRmRNA is evident at 24 h but not at 6 h (Figs. 1 and 2).

A similar time course for VDRmRNA upregulation anddownregulation by 1,25(OH)2D3 has been demonstrated in in-testine (18), whereas in human pomyelocytic leukemia cells(HL60) 1,25(OH)2D3 has been shown to upregulate the VDRmeasured with a monoclonal antibody at 12 h, and down-regulate the VDR at 48 and 72 h (20). Ligand binding studieshave determined that 1,25(OH)2D3 increases VDR number ina number of cell systems (19, 20). Huang et al. (17) studiedduodenal VDRmRNA of vitamin D-deficient rats with andwithout 1,25(OH)2D3 treatment (25 ng/day for 7 d). Theyfound no change in VDRmRNA as measured by slot blots.The differences in the effect of 1,25(OH)2D3 on duodenalVDRmRNA when given as a single injection by Strom et al.(18) and in the present study, as compared to chronic treat-ment ( 17), remain to be clarified.

Parathyroid VDR protein levels and 1,25(OH)2D3 bindingcapacity were not determined in the present study. However,Strom et al. (18) studied rat intestine VDR, and showed that1,25(OH)2D3 lead to an increase in both VDRmRNA andVDR protein. Moreover, Costa and Feldman (16) had shownthat 1,25(OH)2D3 treatment in vivo lead to an increase inVDR binding. Additionally, a prolongation in VDR half-lifewas shown to be a major factor in the VDR upregulation byvitamin D metabolites in vitro studies (30). Therefore1,25(OH)2D3 upregulates its receptor at a number of levels,including transcription, translation, and receptor half-life.

In the present study Northern blots from parathyroid-thy-roid tissue of rats maintained on a vitamin D-deficient diethad a predominant 2.2-kb VDR transcript. In contrast in con-trol rats the 4.4-kb VDR transcript predominated (Fig. 3).

The total parathyroid VDRmRNA was not very differentbetween control rats and rats which had been on a vitaminD-deficient diet, but rather the distribution between 2.2- and4.4-kb transcripts. The interpretation of this finding is notclear, but it might be speculated that the larger transcript is amore stable transcript.

We showed that serum calcium had no effect on parathy-roid-thyroid VDRmRNA levels. Favus et al. (31) showed thata low calcium did not affect duodenal VDRmRNA levels de-spite increasing VDR number, suggesting a posttranslationaleffect. The effects of a low calcium diet on intestinal VDRnumber might be due to the effect of low calcium itself or ofthe increased serum 1,25(OH)2D3 levels in these rats (31). Ratswith normal vitamin D and low calcium (ND-Ca) did not havehigher parathyroid VDRmRNA levels, despite increasedserum 1,25(OH)2D3 levels. This might imply that a normalcalcium is necessary for 1,25(OH)2D3 treatment to upregulatethe VDR gene's transcription. In contrast a rat duodenumextract had a single VDRmRNA band at 4.4 kb, which wasincreased after 1,25(OH)2D3 (Fig. 4), similar to published re-sults. VDRmRNA has been shown to run as more than oneband in mouse 3T6 fibroblasts (8), chicken intestine and brain(8), and rat kidney (17) and as a single band in chicken brain(8) and rat intestine (17). The significance ofthis heterogeneityis not clear.

In the dietary model of secondary hyperparathyroidismdue to a low calcium and deficient vitamin D intake there wasa marked increase in PTHmRNA but no increase inVDRmRNA. In another model of secondary hyperparathy-

roidism, chronic renal failure due to 5/6 nephrectomy, therewas also no increase in VDRmRNA (32). These results indi-cate that secondary hyperparathyroidism does not result inparathyroid cells which are stimulated to synthesize all theirproducts at an increased level, but rather synthesize morePTHmRNA than other cellular products such as VDRmRNA.

The rat's two parathyroids are embedded in thyroid tissueand it is therefore not possible to accurately microdissect theparathyroids from the thyroids without significant contamina-tion with thyroid tissue. For this reason the experiments re-ported here were all performed on rat parathyroid-thyroid tis-sue, which is irrelevant when measuring total rat PTHmRNA,but is relevant when presenting results for VDRmRNA. This isbecause the thyroid parafollicular or C cells which synthesizecalcitonin also have VDRS (33), and calcitonin gene transcrip-tion is also regulated by 1,25(OH)2D3 (6). It was thereforenecessary to determine the relative contribution of parathyroidcell VDRmRNA to C-cell VDRmRNA. We used two experi-mental procedures, separation of the cell populations by flowcytometer (FACS) and in situ hybridization.

For flow cytometry studies a cell suspension from rat para-thyroid-thyroid was separated by size into two peaks whichwere collected separately, and as we have reported previouslythis separates the parathyroid cells from the thyroid follicularcells (Fig. 7) (27). RNA extracts from the first peak of smallercells had PTHmRNA, which was not present in the secondpeak of larger cells, whereas thyroglobulin mRNA was onlypresent in the second peak (Fig. 7). Calcitonin mRNA was alsorestricted to the second peak. VDRmRNA was present in thefirst peak with only a trace amount in the second peak (Fig. 7).This would suggest that quantitatively the VDRmRNA fromparathyroid-thyroid is almost all from the parathyroids.

The second methodology we used was in situ hybridizationfor VDRmRNA (Figs. 8 and 9). Sections from rat parathy-roid-thyroid tissue and duodenum were processed and hybrid-ized under conditions that favor formation of RNA-RNA hy-brids (28, 29). Rat from parathyroid-thyroid tissue from onelobe of a control rat, together with parathyroid-thyroid andduodenum from a rat which had received 1,25(OH)2D3 (100pmol at 24 h) were fixed to a microscope slide together, inorder to allow comparison among the sections. VDRmRNAwas markedly concentrated to the parathyroids as compared tothe thyroids, as well as to the duodenal mucosa (Figs. 8 and 9).The intensity of VDRmRNA was greater in the 1,25(OH)2D3treated rat's parathyroid than in the control. The results of thedot blots and Northern blots presented here therefore repre-sent VDRmRNA derived from parathyroid tissue, much morethan that from the parafollicular cells. Moreover, using pri-mary cultures of bovine parathyroid cells we have demon-strated a similar upregulation ofVDRmRNA by 1 ,25(OH)2D3(data not shown). It remains to be studied whether1,25(OH)2D3 also regulates VDR gene expression in calcitoninproducing cell lines.

1,25(OH)2D3 is a potent regulator of PTH gene transcrip-tion, decreasing PTH gene transcription in vivo by > 90% aftera single injection in the rat (4). We have now demonstratedthat it also increases VDR gene expression in the parathyroidcell, which might result in increased VDR protein synthesisand increased binding of 1,25(OH)2D3. This ligand dependentreceptor upregulation would lead to an amplified effect of1,25(OH)2D3 on the PTH gene, and might help explain thedramatic effect of 1,25(OH)2D3 on the PTH gene.

1974 Naveh-Many, Marx, Keshet, Pike, and Silver

Acknowledgments

We thank Dr. Howard Cedar for helpful discussions, Dr. H. Mayer forthe PT43 plasmid, Dr. M. J. Rosenfeld for the rat calcitonin gene, Dr.Vassart for the thyroglobulin gene, Drs. Kaiser and Eischer of Hoff-man-La Roche Inc., Basel, Switzerland, for the 1,25(OH)2D3, Ms. M.Ofner and A. Itin for technical assistance; and Dr. H. Gilo and Ms. G.Neiman for help with the FACS.

This work was supported by grants from the National Institutes ofHealth (grant DK 38696), the United States-Israel Binational ScienceFoundation (BSF), the German-Israel Foundation for Research andDevelopment (GIF), and the Israel Academy of Sciences and Human-ities.

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1,25(OH)2D Receptor Gene Expression 1975